Protein C and protein S deficiencies contribute to morbidity and mortality in men and mice. Recombinant activated protein C (APC) therapy reduces mortality in adult severe sepsis patients. There is a major need for new insights into the physiologic and pharmacologic mechanisms of action of APC and protein S. To establish in vivo proof of principle for such mechanisms, this project uses genetically modified mice, murine injury models, and novel recombinant murine proteins. APC can exert two major, distinct activities: (1) anticoagulant activity and (2) direct beneficial effects on cells comprising a variety of cytoprotective actions. This latter activity is critical for mortality reduction by APC in murine sepsis models. The current paradigm for APC's cell signaling involves binding of APC by endothelial protein C receptor (EPCR) combined with protease activated receptor-1 (PAR1) proteolytic activation. We found that there is another signaling pathway initiated by APC that involves ligation of apolipoprotein E Receptor 2 (apoER2), signaling via the adaptor protein, Dab1, and Src-family kinases with downstream activation of the PI3K-Akt survival pathway. Engineering of murine APC and apoER2 mutants will allow interrogation of the protein surfaces that mediate binding and signal initiation by APC:apoER2 interactions and will provide reagents for in vivo proof of principle studies for mechanisms of APC's action in murine sepsis. Studies of mice genetically modified in apoER2 and Dab1 will establish whether apoER2 and Dab1 mediate APC's mortality reduction activities in sepsis. Protein S deficient mice will be subjected to thrombotic provocation and treated with combinations of recombinant wild type and mutant murine protein S and APC or other agents to define the relative efficacies for protein S antithrombotic activity that is either dependent on APC or independent of APC. Novel Principles that are established by these preclinical animal model studies may ultimately be translated into diagnostic or therapeutic advances involving the protein C and protein S systems. PUBLIC HEALTH RELEVANCE: Previous basic research on plasma Protein C, a naturally occurring plasma protein, was translated into diagnostic and therapeutic tools now used in the clinic. The proposed basic research studies on activated protein C will provide proof of principle and definitive insights into molecular mechanisms by which activated protein C acts on blood cells to prevent damage that can be fatal. The findings may well be translatable into future clinical advances.